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SAFETY EVALUATION OF TOOTHPASTE CONTAININGCHLOROFORM III. LONG-TERM STUDY IN BEAGLE DOGS

R. Heywood, R. J. Sortwell, P. R. B. Noel, A. E. Street, D. E.Prentice, F. J. C. Roe, P. F. Wadsworth, A. N. Worden

Huntingdon Research Centre, Huntingdon, England.

N. J. Van AbbeMedical Affairs and Safety Evaluation Unit, Beecham Products,Research Department, Randalls Road, Leatherhead Surrey, England

Beagle dogs were given chloroform in a toothpaste base orally ingelatin capsules on 6 d/wk for 7 1/2 yr, followed by a 20-24 wkrecovery period. Groups of 16 males and females received 0.5ml/kg/d of the vehicle (toothpaste without chloroform) and 8dogs of each sex remained untreated. Treated groups comprised 8dogs of each sex, receiving doses equivalent to 15 and 30 mgCHCI3/kg/d in the toothpaste vehicle; another group of the samesize received an alternative non-chloroform toothpaste (0.5ml/kg/d). Eleven of the 96 dogs died during the study, only two ofthese being in the CH03-treated groups. The only significanttoxic response during treatment was a moderate rise in serumenzyme levels (e.g. SGPT), reaching a peak in the sixth year ofthe study and probably corresponding to minimal liver damage.Few palpable growths were noted while the dogs were alive."Fatty cysts" were seen in the liver of several dogs at postmortem possibly associated with the chloroform treatment but thedistribution of a nodular change in the liver was not obviouslydose related. A small number of macroscopic and microscopicneoplasms were seen; one dog in each chloroform-treated grouphad a malignant tumour but there were no tumours in the liver orkidney of any dog. Overall, exposure to chloroform in a tooth-paste base was not associated with any effect on the incidence ofany kind of neoplasm.

From this and related studies in mice and rats, it is conclud-ed that repeated exposure to chloroform (3.5 percent) in tooth-paste is unlikely to result in any hazard to human health.

INTRODUCTIONStrain-related differences in renal tumour incidence in mice given

toothpaste containing chloroform for 80 wk (Roe, et al., 1978) suggestedthe need for a carcinogenicity study using a non-rodent species. At thetime concerned (1967), guidance was given by the U.S. Food Protection

Please send requests for reprints to N. J. Van Abbe.

Journal of Environmental Pathology and Toxicology 2:835-851Copyright © 1979 by Pathotox Publishers, Inc.

836 HEY'WOOD , ET AL.

Committee (1959) recommendation for a 4-yr dog study. Interest in thedog stemmed from the well-known finding of Hueper, et al., (1938) thatbladder tumours in dogs given 2-naphthylamine required a latent periodof only about 2 yr; such tumours could not be demonstrated in rats ormice though they did occur in man. Several later reviews discussed theuse of dogs for carcinogenicity screening, e.g. Berenblum (1969), Bonser(1969) and Lalonde et al. (1973).

The present report deals with preliminary studies for the selection ofdose levels and the main study, which was planned to continue for 7 yr.

MATERIALS AND METHODSAnimal Management and TreatmentFor the main study, 48 male and 48 female pure bred beagles,

initially 18-24 wk old, were housed singly in kennels. Another 8 malesand 8 females were used for preliminary studies. All the dogs wereclinically examined and inoculated against distemper, canine hepatitisand leptospirosis as well as being dosed with piperazine adipate as ananthelmintic. Inoculation and anthelmintic treatment were repeated an-nually.

The dogs were fed weighed amounts (200 g) of a dry diet at fixedtimes twice daily. When new food was offered, any residue from theprevious meal was removed for weighing and calculation of food con-sumption. From week 300, the daily food ration of dogs considered to beobese was reduced from 400 to 300 g. Water was freely available at alltimes and fresh milk (200 ml) offered to each dog on weekday morningsfor the first 6 mon.

The test material given to the dogs was a toothpaste complying withthe formula given by Roe et al., (1978) except that the carragheen gumand glycerol concentrations were reduced for ease of transfer from asyringe into gelatin capsules. Transfer was effected immediately beforedosing, to minimize volatilization of chloroform or softening of thegelatin. Doses, calculated weekly after weighing the individual dogs, weregiven at a-fixed dose-volume of 0.5 ml/kg on 6 d/wk.

For humane reasons, treatment e.g. with antibiotics was given to afew dogs with obvious illness in the late stages of the main study. Avehicle control bitch developed a large mammary growth which wassurgically removed during the final year and another in the sametreatment group was operated upon for the repair of an inguinal herniaduring the 5th year of the study.

Dose-Levels (Preliminary Study)

The schedule of treatment given 7 d/wk is shown in Table 1. At thetime of commencing these studies, reference data on untreated beagleswere not readily available through Brunk (1969), Noel (1970), Deavers etal., (1972) and Lutzen et al., (1976) have since given details. Data were,

LONG-TERM STUDY OF CHLOROFORM IN DOGS 837

however, available to us from another 64 beagles of similar age from thesame supplier.

Dose-Levels (Main Study)

From the observations made in the preliminary study and to ensurethat the upper dose level would reach the threshold for early toxicchanges but with as many dogs as possible remaining alive and well forat least 7 yr, daily dose-levels of 0, 15, and 30 mg CHCI 3/kg wereselected for the main study. The treatments are detailed in Table 2.

Observations

A full range of data was collected during the preliminary study andterminal histopathology was carried out. In the main study, clinical signswere recorded daily, food consumption twice daily, water consumptionintermittently and bodyweight once per week. Ophthalmoscopy was

TABLE 1. Treatments given in Preliminary Study

No. of dogs Treatment' Duration(mg CHCI3/kg/d) (weeks)

1 1 120 12

1 1 90 12

2 2 60 18

2 2 45 13

2 2 30 13

1 In toothpaste base (including peppermint oil and eucalyptol) given orally bygelatin capsule.

performed on each dog once during the pre-treatment period and then at3-month intervals after the start of treatment. Each dog had a thoroughclinical examination at least twice yearly. A comprehensive range oflaboratory investigations was performed on all dogs once during thepre-treatment period and then, after treatment started, at 6 and 13 wk, 6,9and 12 mon and thereafter at 6-mon intervals. Additional serum enzymestudies were undertaken in the later stages of the investigation to monitorliver status.

Post-Treatment Recovery Phase and Terminal Studies

As 84 of the original 96 dogs were still alive and in good conditionafter 7 yr, the intention to end the study at this stage was reconsidered.The only sign of toxicity was a moderate elevation of certain serumenzyme levels, mainly at the higher chloroform dose-level; there were nopalpable tumours but a few subcutaneous nodules thought to be ofmammary gland origin were detectable.

838 HEYWOOD , ET AL.

Continuation of the experiment would have allowed further time forpossible treatment-related lesions to develop, but it was thought better tosacrifice all the animals at one time so that strictly valid comparisonscould be made between different groups. The dogs were, however, keptunder observation for several months after the cessation of treatment atweek 376 to see whether the elevated serum enzyme levels reverted tonormal.

Between weeks 395 and 399, each dog still alive received anintravenous injection of sodium pentobarbitone. Immediately after killingor as soon as possible, in the case of animals found dead, full macro-

TABLE 2. Treatments given in Main Study.

No. of dogs Treatment(mg CHCI3/kg/d)

8 30a

8

8 15a

16 16 0a a,b

8 8 Untreated

8

8 Alternative non-chloroform toothpaste

a ln toothpaste base (including peppermint oil and eucalyptol) givenorally by gelatin capsulebVehicle Control Groupcrhe same alternative non-chloroform toothpaste referred to by Roeet al. (1978)

scopic examination was made of all tissues and any abnormalities noted.The principal organs were then removed and weighed; small portions ofthese and a wide range of other tissues along with portions of any lumpor tumour were placed in fixative. Sections were routinely stained withhaematoxylin and eosin; in addition, liver and kidney sections werestained with Oil Red 0 for fat. Further examination, by electron micro-scopy, was conducted terminally on liver and kidney sections from twountreated control dogs and three dogs from the 30 mg CHCI 3/kg/d group.

RESULTSPreliminary StudyNo dogs died during the preliminary study. Vomiting occurred at

times in dogs given 120, 60 or 30 mg CHCI 3/kg/d and one dog that re-

LONG-TERM STUDY OF CHLOR0fORM IN DOGS 839

ceived 120 mg CHCI3/kg/d became jaundiced after treatment for 4-5weeks. Loss of general condition was noted in both dogs at 120 mgCHC13/kg/d, in the male at 90 mg CHCI 3/kg/d and in one dog of eachsex at 60 mg CHCI 3/kg/d. Marked bodyweight losses occurred in the dogsgiven 60 mg CHCI3/kg/d or above and some others had reduced weightgain. Appetite was apparently suppressed at first in all dogs and not re-gained in dogs which showed effects on bodyweight.

Increase in SAP, SGOT, bilirubin and ICD was seen in the male at120 mg CHCI3/kg/d. An indication of the SGPT values found is given inTable 3. The female given 120 mg CHCI 3/kg/d and both dogs given 90mg CHCI3/kg/d showed increased SGPT on several occasions and bili-

TABLE 3. SGPT Values in Preliminary Study'

Treatment(mg CHCI 3 /kg/d): Untreated 30 45 60 90 120

Sex: dYd 9 d 9 d 9 d 2 d 9

Mean SGPT 2

(mU/m1): 28 29 68 45 50 69 92 58 84 114 146 47

1 At 12 wk for treated dogs, 5 wk for untreated controls.2 32 males and 32 females in Untreated group; 2 males and 2 females in 30, 45 and 60 mg CHCI3/kg/dgroups, other data are for single dogs.

rubin once but no other definite abnormality. There was an increase inSGPT at the dose level of 60 mg CHCI 3/kg/d and, in the two dogsshowing marked loss of condition, an effect on SAP and SGOT. Onlyoccasional increases in SGPT were seen at 45 and 30 mg CHCI 3/kg/d. Atpost-mortem there was discolouration of the liver and increased liverweight at 45 mg CHCI3/kg/d and above. Nothing abnormal was seen at30 mg CHCI 3/kg/d. At 60 mg CHCI 3/kg/d and above, hepatocyte enlarge-ment and vacuolation together with deposition of fat within the hepato-cytes was seen in all dogs. Variation in hepatocyte size with slight fatdeposition was noted at 45 mg CHCI3/kg/d.

Main Study

Shortly after the study began, frequently observed changes were softand pale faeces along with occasional vomiting in all treated groups andin the vehicle controls. Regular veterinary examination revealed noevidence of disease apart from transient minor abnormalities and one caseof inguinal hernia in a vehicle control bitch, none of which weretreatment related.

840 HEYWOOD , ET AL

About 20 percent of the dogs were hyperexcitable, especially duringthe first 2-3 yr. These dogs were prone to convulsive episodes andaltogether 10 dogs died after one or more fits. A vehicle control bitch inrenal failure was killed in week 299 for humane reasons; this dog wasapparently in good health for the first 51/2 years but then deterioratedrapidly, showing various degenerative tissue changes at post mortemwithout obvious cause. Deaths are listed in Table 4. During the final year,a bitch from the group receiving the alternative non-chloroform toothpastewas missing after kennel cleaning and not subsequently found.

Bodyweight changes are summarized in Figs.- 1 and 2 for males andfemales respectively still alive at the time of weekly weighing. Thefindings were complicated by obvious obesity; dogs considered obese,whose diet was reduced at the beginning of week 300 are listed in Table

TABLE 4. Deaths during Main Study

Treatment No. of Deaths Wk. of Death

(mg CHCI 3/kg/d) d 9

30 1 0 87

15 1 0 328

Vehicle Control 1 4 96, 125, 151, 173, 299a

Untreated 1 3 134, 147, 153, 192

Alternative non-chloroform toothpaste 0 0

a Killed for humane reasons.

5. Neither the development of obesity in individual dogs nor the groupmean bodyweight changes were treatment related. Food consumption wasnot influenced by the treatments given. Males consistently left not morethan 5 percent of their food but females regularly left up to 15 percent.Daily water consumption was in the region of 1000-1500 ml for all dogs.

Haematological findings during the study and at the end of treatment(week 374) showed no discernible differences between groups. The mostobvious deviation found in biochemical analyses was a dose-relatedelevation in SGPT values, later accompanied by less marked elevation ofSAP and SGOT. No other parameters originally studied showed anyabnormality in the treated dogs. Since moderate liver damage wassuggested by the analytical findings, bromsulphthalein retention tests wereperformed during the sixth year of the study but no influence due totreatment was detected. Later the biochemical analyses were extended toinclude further indices for presumptive liver damage, namely LAP, yGT

LONG-TERM STUDY OF CHLOROFORM IN DOGS 841

and GDH. Tables 6 and 7 show indications of hepatic dysfunction whiletreatment continued; however, all but one of the dogs at the lowerCHCI3-dose level and several of the higher dose-level dogs reverted tonormal SGPT values during the post-treatment recovery phase, whereasseveral dogs in other groups had increased SGPT values at this stage.Changes noted in ophthalmoscopic examination included peripheral cap-sular opacities associated with the ends of posterior lens sutures, a degreeof nuclear sclerosis and some degenerative change involving the tapetum

—.30mg CHC13/Kg/d—15rng CHCIA1c1--- Vehicle Control— Alternative non-CHC13 toothpaste-- Untreated

1 12 36 dO 84 168 132 166 'A0 264 218 266 2t6 360 314 348 3t6 420Treatment Weeks

FIGURE 1. Group Mean Bodyweight (Males)

lucidum; none of these age-related changes was considered to be treat-ment-related. There were few anomalies in urinalysis at the end oftreatment or at any intermediate stage.

Autopsies on the dogs that died during the treatment phase of thestudy showed no obvious changes in the brain, other parts of the centralnervous system, liver, kidney or other organs and there were no tumours.

At the end of the study there were no treatment-related differences ineither absolute or relative weights of various organs (Table 8); consider-able individual variations were noted butgroup means showed nosignificant deviations when compared with the untreated or vehiclecontrols.

Histopathological Findings Other Than NeoplasmsLiver and Gall Bladder. Generalised disruption of lobular architecture

was seen in two of the upper dose-level males and one male vehiclecontrol. Varying incidence of bile duct hyperplasia, either focal or

Diet restrictionfor obese dogs

commenced

End oftreatment

......

41: .../ • _ _

................. ......

-30mg CHCI3/Kg/d-15mg CHCI3/Kg/d--- Vehicle Control Alternative non-CHCI3 toothpaste- Untreated

12 36 60 84 108 132 156 180 204 228 256 275 300 324 348 376 420. 11 I 1 u

Treatment Weeks

20•

t

7:15

10•

842 HEYWOOD , ET AL

generalised, was seen in all groups without any clear relationship totreatment; similarly, the frequency of minimal hepatic fibrosis was notdose-dependent. Aggregations of vacuolated histiocytes forming so-called"fatty cysts" (Jubb and Kennedy, 1970) were seen in all groups, althoughthe cysts were larger and more numerous in the treated dogs (Table 9).

Haemosiderin, almost entirely within macrophages, was found in thelivers of all dogs but most prominently in the group treated with thealternative non-chloroform toothpaste (40 percent of group with haemosi-derin in numerous macrophages; highest in other groups was 11 percentof dogs in the vehicle controls). Twelve dogs had small areas of

FIGURE 2. Group Mean Bodyweight (Females)

well-defined nodules of altered hepatocytes, in seven of which onlymicroscopic nodules were detectable. The nuclear morphology of thealtered hepatocytes was normal; no mitoses were seen and the areasappeared to be early or small hyperplastic nodules without evidentneoplasia. Dogs in all groups showed these changes, which had noobvious relationship to treatment. Numerous minor hepatic changes andminor abnormalities of the gall bladder were seen, apparently unrelated tothe treatment given.

Cardiovascular System. Small focal areas of arteriosclerosis wereseen in the aorta of about one-half of the dogs in each group andendocardiosis of the mitral and/or tricuspid valves in a small proportion ofdogs from all groups. These and other less marked changes did not appearto be treatment-related.

LONG-TERM STUDY OF CHLOROFORM IN DOGS 843

Reproductive System. Ectopic testes with inhibition of spermatogene-sis were noted in two dogs at the upper chloroform dose level, one at thelower dose level and one untreated control. Other abnormalities in malesoccurred in various groups without any treatment relationship. Most of thechanges in the ovaries and uterus of the females were obviously consis-tent with normal oestrus activity. Nodular hyperplasia of the mammarygland was noted in three dogs at 15 mg CHCI 3/kg/d, five of the vehiclecontrols and one untreated control.

Urinary System. Prominent changes, usually chronic interstitial neph-ritis, were seen in the kidneys in all groups but most often in theuntreated controls (4/12). Fat deposition accounted mainly for the differ-ences between groups in glomerular abnormalities. Many glomeruli wereaffected in 6/15 dogs of the 30 mg CHCI 3/kg/d with fewer glomeruliaffected in all other groups.

Other Systems. Numerous changes were recorded in individual dogs,without evident treatment relationships.

TABLE 5. Dogs Considered Obese at Wk 300

Treatment No. of Obese Dogs(mg CHCI3/kg/d) a 9

30

2

6

15

2

8

Vehicle Control

7 9

Untreated

4

Alternative non-chloroform toothpaste 3 5

Histopathological Findings: NeoplasmsNo neoplasms were seen in dogs which died during the study. The

details for neoplasms found in dogs killed at the end of the study aregiven in Table 10. There was no evidence of invasion of other tissues byany of the tumours, although two were classified as histologicallymalignant.

DISCUSSIONThe prevalence of a type of idiopathic epilepsy (Koestner and Rehfeld,

1968; Martinek and Dahme, 1977) in the early stages of our main studyraised doubts about the feasibility of achieving the projected 7-yr term butfortunately, after the population of most excitable animals had died as a

844 HEYWOOD , ET AL.

result of fits, there were no more deaths from this cause. However, theoptimal duration for a carcinogenicity study using dogs is still debatable.Compared to many dog studies terminated after only 2 to 4 yr, the workreported here provides more comprehensive long-term findings, and itcompares favourably also with earlier studies lasting 9 or more years withonly two or three dogs per treatment. Recently, Weikel and Nelson (1977)reported a 7-yr study of contraceptive steroids given to substantialnumbers of female beagles.

TABLE 6. Group Mean Values in Biochemical Analyses (Main Study) (males and females combined)

Treatment(mg CHCI3/kg/d)

30 15

Alternative

Vehicle Un- non-

Control treated chloroformtoothpaste

No. of dogs 15 15 27 12 15

Wk. 374a 395b 374 395 374 395 374 395 374 395

Urea (mg %) 30 28 32 26 31 31 33 27 34 29

Glucose (mg %) 99 99 100 100 101 98 99 95 102 98

Total serum proteins (g%) 5.9 6.1 6.1 6.2 6.3 6.0 6.3 6.0 6.4 6.1

Albumin 2.9 2.8 3.0 2.8 3.1 2.8 3.2 2.9 3.2 2.9

al globulin 0.3 0.3 0.3 0.3 0.3 0.4 0.3 0.3 0.3 0.3

a2 globulin 0.7 0.8 0.7 0.8 0.7 0.8 0.7 0.7 0.7 0.8

f3 globulin 1.6 1.7 1.7 1.7 1.7 1.6 1.6 1.6 1.7 1.6

y globulin 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.4 0.6 0.4

A/G ratio 0.98 0.86 0.98 0.87 0.99 0.91 1.04 0.97 1.00 0.94

SAP (KA units) 30 26 20 17 14 16 12 14 10 13

SGPT mU/m1 102 111 66 48 51 128 50 56 57 87

SGOT mU/m1 33 33 27 29 25 32 22 32 25 31

LAP (GR units) 90 72 82 62 73 54 81 56 82 60

Bilirubin (mg %) <0.2 <0.2 <0.3 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2

Erythrocyte cholinesterase(A pH/h) 0.73 0.73 0.78 0.81 0.77 0.81 0.77 0.82 0.65 0.71

Plasma (A pH/h) 1.08 0.91 1.00 0.88 1.00 0.87 1.02 0.86 1.05 0.91

y GT (mU/m1) 3.6 3.3 2.9 2.5 2.9 2.1 2.5 1.7 2.8 1.8

GDH (mU/m1) 7.2 5.3 6.5 3.9 4.9 4.0 3.9 3.0 4.9 3.0

ICD (B & B units) 11.8 11.4 11.2 10.7 9.9 12.9 9.2 11.7 10.6 12.9

a End of Treatment.

bEnd of post-treatment recovery period.

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TABLE 8. Organ Weights in Main Study(Upper Figure = Absolute wt. in g.; Lower Figure = Organ wt. as % body wt.)

Treatment 30 15 Vehicle Untreated Alternative

mg CHCI3/kg/d Control non-chloroformtoothpaste

77Brain 79 76 81 78

0.66 0.62 0.65 0.64 0.59

Pituitary (m g) 81 77 79 73 79

0.00068 0.00063 0.00063 0.00059 0.00060

Spinal Cord 20.4 19.2 20.3 19.8 20.2

0.17 0.16 0.16 0.16 0.15

Heart 114 118 121 117 120

0.94 0.95 0.96 0.95 0.90

Lungs 107 108 112 105 116

0.89 0.87 0.90 0.85 0.88

Liver 473 470 433 462 451

3.86 3.82 3.47 3.74 3.42

Spleen 88 75 71 63 65

0.71 0.60 0.56 0.50 0.49

Pancreas 34 40 35 37 39

0.28 0.32 0.28 0.31 0.29

Thymus 2.4 2.5 - 2.8 3.7 2.9

0.02 0.02 0.02 0.03 0.02

Prostate 22.5 21.8 23.8 20.1 21.0

0.16 0.17 0.18 0.16 0.16

Uterus 11.7 8.5 10.0 13.0 11.3

0.11 0.07 0.08 0.10 0.09

Kidneys 78 79 78 84 78Kid

0.64 0.64 0.62 0.68 0.59

Thyroids 0.96 0.86 0.97 0.91 0.81

0.0080 0.0071 0.0077 0.0073 0.0061

Adrenals 2.23 2.23 2.06 2.05 2.09

0.0186 0.0183 0.0164 0.0170 0.0158

Testes 34.0 30.6 31.0 22.6 30.2

0.236 0.232 0.243 0.209 0.233

Ovaries 1.32 1.57 1.45 1.82 1.67

0.0120 0.0133 0.0119 0.0143 0.0124

Body-weight 12350 12420 12730 12510 13340

LONG-TERM STUDY OF CHLOROFORM IN DOGS 847

The upper dose level selected for our main study was based on therelatively small changes in SGPT levels during the preliminary study,since hepatotoxicity was expected to be a determining factor. This para-meter evidently served the purpose well in this instance, and we werealso able to include a lower dose level representing approximately onehundredfold exaggeration compared to normal human exposure.

We were reluctant to undertake any test procedure during the mainstudy that might have prejudiced its long-term outcome. Thus we onlyperformed limited BSP studies to assess liver function. As in the prelimin-ary study, the most useful index of relatively low-level hepatic dysfunction

TABLE 9. Liver Changes (Nodules of Altered Hepatocytes and "Fatty Cysts")

No. of dogs No. with "fatty cysts"Examined his-

Treatment Sex tologically at No. with Occasional Moderate(mg CHCI3/kg/d) end of expt. nodules or minimal or marked

30 d 7 0 1 6

8 4 0 7

15 d 7 1 0 6

8 1 2 3

Vehicle Control d 15 0 7 1

12 3 3 0

Untreated d 7 1 2 0

5 1 1 0

Alternative d 8 0 2 0non-chloroformtoothpaste 9 7 1 0 0

during the full -7-yr term was the modest elevation of SGPT levels, whichrose steadily during the first 5 yr and later gradually declined. The mostprominent histopathological change in the liver was the occurrence offatty cysts. However, whereas SGPT tended to revert to normal levelsduring the post-treatment recovery phase, the fatty cysts persisted andmay, therefore, have been unrelated to the enzyme changes. In man, theonset of liver damage is often accompanied by changes in ICD levels(Tietz, 1976), but no significant deviations from normal values wererecorded in our study.

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Taking microscopically-discernible tumours into account as well asmacroscopic lesions, the duration of our study was clearly sufficient toreach the time at which spontaneous tumours begin to appear in thebeagle dog. Since there was no dose-dependent increase in tumours atthis stage related to the treatment given, the treatment clearly did notadvance their onset. Of the tumours recorded in our study, a few werefound on microscopic examination alone. At the 71/2-yr stage in thelifespan of the beagle, tumours of the testis and mammary gland wereevidently starting to devlop rather commonly but we saw no reason toattribute malignancies to the treatment given. Above all, there was acomplete absence of neoplasia affecting the liver or the kidney, eventhough the upper dose-level was high enough to exert some degree ofhepatotoxic action and some of the dogs in all groups developedhyperplastic liver nodules.

To summarise the position with regard to the safety evaluation oftoothpaste containing up to 3.5 percent of chloroform, data now existfrom long-term studies in mice, rats and dogs. In mice, though only at thehigher dose-level given, there was a sex- and strain-related tendency todevelop renal tumours predominantly benign in character. By contrast, atdose-levels up to 400 times the estimated human exposure to chloroformfrom twice daily toothbrushing, we did not observe any overall increasein neoplastic changes in male mice of three of four strains, in female miceor in rats; at dose levels up to 200 times estimated human exposure, notreatment-related excess of tumours was observed in beagle dogs afternearly 71/2 yr. Eschenbrenner and Miller (1945) showed that chloroformgiven in vegetable oil solution was carcinogenic to the liver of femalemice but only when given in doses large enough to produce liver necrosisand to kill all the treated male mice. The NCI report (1976) also dealtwith the effects of high dose-level administration of chloroform invegetable oil solution, noting an excess of liver tumours in mice and renaltumours in rats. We have shown that the findings in chloroform studiesmay be markedly influenced by administration in vegetable oil solutionrather than toothpaste. Recently Butterworth (1978) confirmed the tenden-cy for [ 14C] chloroform to concentrate radioactivity in the kidneys of malemice when given as a solution in a vegetable oil, but he found nosignificant radioactivity except in the urinary bladder 1 hr after giving thesame chloroform dose in a toothpaste vehicle to male MF1 mice.Moreover Brown et al. (1974) demonstrated marked differences betweenthe metabolism of chloroform given to rodents and a non-rodent (thesquirrel monkey).

The range of experimental evidence now available appears to befully consistent with our view that repeated exposure to chloroform at alevel of 3.5 percent in toothpaste is unlikely to result in any hazard tohuman health.

LONG-TERM STUDY OF CHLOROFORM IN DOGS 851

REFERENCES

Berenblum, I.: Carcinogenicity Testing. UICC Tech. Report Series, Vol. 2, 1969.Bonser, G. M.: How Valuable the Dog in the Routine Testing of Suspected Carcinogens. J. Nat. Cancer

Inst. 43:271, 1969.Brown, D. M., Langley, P. F., Smith, D. and Taylor, D. C.: Metabolism of Chloroform-I. The

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JEPT MS 419CReceived June 20, 1978

Accepted September 6, 1978